Proceedings of the 41st Annual ASTRO Meeting 295

evaluate the effect of rMETase on intracellular GSH levels in H-460 cells. We are further investigating additional cell lines and in vivo models with the intent of developing rMETase as a clinical radiation sensitizer. As we have already constructed several viral gene therapy vectors carrying rMETase, we are evaluating them for radiation sensitization capability.

2033 COMPARISON OF CONTINUOUS LOW DOSE RATE AND PULSED DOSE RATE BRACHYTHERAPY IN AN ANIMAL TUMOR MODEL

Harms W’, Peschke P’, Weber K3, Hensley p, Wannenmacher M5

UniversiQ Heidelberg, Heidelberg, Germany’; German Cancer Research Center, Heidelberg, Germany’; University Heidelberg, Heidelberg, Germany’; University Heidelberg, Heidelberg, Germany”; University Heidelberg, Heidelberg, Germany’

Purpose: Theoretical calculations suggest that PDR should have the same effectiveness as CLDR brachytherapy. The aim of this study was to compare the effectiveness of CLDR and PDR brachytherapy in an animal tumor model.

Methods: Interstitial PDR and CLDR brachytherapy was administered to the Dunning prostate R3327-AT1 carcinoma transplanted subcutaneously into the thigh of Copenhagen rats. A dose of 20, 30, 40 and 50 Gy was administered in each study arm. 8 animals were irradiated per dose group and technique. The dose was prescribed to the tumor surface (5 mm source distance, tumor diameter 10 mm). Interstitial PDR was carried out using a 37 GBq 1921r source with 0,75 Gyipulse and hour. CLDR was administered with a centrally implanted seed with a dose rate of 0,75 Gy/h. Endpoint was growth delay. Each rat was observed for 120 days. The tumor size was measured three times a week. Treatment response was judged on the basis of the time (TS/days) required for each tumor to reach 5 times the initial tumor volume. Flow cytometry and immunochemistry were carried out after 4, 12, 24, 48, 72 and 96 hours and are still under evaluation.

Results: The median T5 times for the CLDR group (20, 30, 40, 50 Gy) were 54.5, 64.5, 85.5 and 65 days. The corresponding T5 times for the PDR group were 21, 3 1, 18.5 and 63 days. Comparison of median values (Wilcoxon Test) showed statistical significance for the lower dose points (20-40 Gy, p=O,OO25).

Conclusion: In this tumor model this preliminary data indicate that PDR brachytherapy seems to be less effective than CLDR in the dose range from 20-40 Gy.

2034 ADENOVIRAL-MEDIATED EZF-1 EXPRESSION SENSITIZES PROSTATE CANCER CELLS IN VITRO TO IONIZING RADIATION

Salem N, Hunt K, Meistrich ML. Meyn R, Pollack A

lJ.T.44.D. Anderson Cancer Center, Houston, TX, USA

Purpose: The transcription factor E2F-1 regulates cell cycle progresion in the Gl and S phases and is implicated in the DNA damage response pathway. The overexpression of E2F-1 causes apoptosis that appears to be independent of p53 status. Preliminary results indicate that E2F-1 overexpression may also sensitize cells to radiation. In this report, the effects of transgene E2F- 1 expression on the clonogenic survival of prostate cancer cells after irradiation was examined. Two cells lines were used; the LNCaP cells express wild type (wt)-p53, while PC3 cells are null ~53.

Materials and Methods: An adenovirus-5-E2F-1 (Ad5-E2F) vector with a CMV promoter was used to generate E2F transgene expression. An AdS/CMV/polyadenylation sequence vector (AdS-pA) was used as a control. The viral solutions were used at a multiplicity of infection of 10. Monolayer cultures (100 ml plates) of LNCaP or PC3 cells were exposed to virus and incubated for 24 hr prior to irradiation using a cesium source. After irradiation to 2, 4, or 6 Gy, the cells were trypsinized, counted, replated, and incubated at 37 C for 14 d. Resultant colonies of 20 cells were counted after fixation and staining.

Results: The plating efficiency in the LNCaP cell line dropped from 41.3% to 1.9% using Ad5-E2F without irradiation. For the PC3 cell line, the plating efficiency was not altered by Ad5-E2F, remaining at approximately 80%. The AdS-pA control did not affect the plating efficiency, with or without radiation, and therefore, is not discussed further. Table 1 shows the results Ad5-E2F and radiation treatment on the clonogenicity of LNCaP and PC3 cells, corrected for the drop in plating efficiency from the virus alone. The LNCaP cell line was exquisitely sensitive to radiation in the presence of E2F transgene expression. The combination at 2 Gy reduced clonogenicity in LNCaP cells by over 30 fold, as compared to 2 Gy in the absence of E2F. In contrast, the combination at 2 Gy reduced clonogenicity by 1.8 fold in PC3 cells. Although the LNCaP cells were more sensitive to the E2F transgene expression and radiation, PC3 cells exhibited significant radiosensitization from E2F.

Conclusion: Transgene E2F expression using an Ad5-E2F vector caused pronounced sensitization of prostate cancer cells to radiation. This effect was most dramatic in LNCaP cells, which express wt-p53. The sensitization of null-p53 PC3 cells was much less, but was still significant. Whether p53 status contributes directly to these differences is not clear.

Table 1. Clonogenic survival after radiation with and without prior treatment with E2F-1. The percentages of surviving clones after 2, 4, and 6 Gy are shown.

2 GY 4 GY 6 GY

LNCaP:E2F- LNCaP:E2F+ 13.7% 0.5%

1.6% 0.0% 0.2% 0.0%

PC3:E2F- 30.2%

8.2% 2.3%

PC3:E2F+ 16.8%

2.4% 0.2%